Why do antimicrobial agents become ineffectual?

Antibiotic resistance has evolved over the past 50 years from a merely microbiological curiosity to a serious medical problem in hospitals all over the world. Resistance has been reported in almost all species of gram-positive and -negative bacteria to various classes of antibiotics including recently developed ones. Bacteria acquire resistance by reducing permeability and intracellular accumulation, by alteration of targets of antibiotic action, and by enzymatic modification of antibiotics. Inappropriate use of an antibiotic selects resistant strains much more frequently. Once resistant bacteria has emerged, the resistance can be transferred to other bacteria by various mechanisms, resulting in multiresistant strains. MRSA is one of the typical multiresistant nosocomial pathogens. A study of the PFGE pattern of endonuclease-digested chromosomal DNA showed that MRSA of a few clones were disseminated among newborns in the NICU of a Japanese hospital. In this regard, it is important to choose appropriate antibiotics and then after some time, to change to other classes to reduce the selection of resistant strains. Since the development of epoch-making new antibiotics is not expected in the near future, it has become very important to use existing antibiotics prudently based on mechanisms of antibiotic action and bacterial resistance. Control of nosocomial infection is also very important to reduce further spread of resistant bacteria.     

Combination antibiotic susceptibility of biofilm-grown <Emphasis Type="Italic">Burkholderia cepacia</Emphasis> and <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> isolated from patients with pulmonary exacerbations of cystic fibrosis

We identified double and triple antibiotic combinations effective against biofilm-grown Burkholderia cepacia and Pseudomonas aeruginosa sampled from cystic fibrosis (CF) patients undergoing acute pulmonary exacerbations. Sputum bacteria from 110 CF patients were grown as biofilms. Combination antibiotic susceptibility testing was used to test 94 double and triple antibiotic combinations. Biofilm-grown bacterial isolates were less susceptible to antibiotic combinations compared to the same bacterial isolates grown planktonically (P < 0.001). Fifty-nine percent of biofilm-grown B. cepacia isolates and 29% of P. aeruginosa isolates were resistant to all double antibiotic combinations tested. Triple antibiotic combinations were more effective than double antibiotic combinations against biofilms (P < 0.0001). For P. aeruginosa biofilms, the addition of azithromycin or rifampin to otherwise effective antibiotic combinations was frequently associated with antagonism. Bacterial biofilms of CF organisms are highly resistant to antibiotics. This study identified potentially effective antibiotic combinations to guide the empirical treatment of CF pulmonary exacerbations.     

Combating antimicrobial resistance: antimicrobial stewardship program in Taiwan

Multi-drug-resistant organisms are increasingly recognized as a global public health issue. Healthcare-associated infection and antimicrobial resistance are also current challenges to the treatment of infectious diseases in Taiwan. Government health policies and the health care systems play a crucial role in determining the efficacy of interventions to contain antimicrobial resistance. National commitment to understand and address the problem is prerequisite. We analyzed and reviewed the antibiotic resistance related policies in Taiwan, USA, WHO and draft antimicrobial stewardship program to control effectively antibiotic resistance and spreading in Taiwan. Antimicrobial stewardship program in Taiwan includes establishment of national inter-sectoral antimicrobial stewardship task force, implementing antimicrobial-resistance management strategies, surveillance of HAI and antimicrobial resistance, conducting hospital infection control, enforcement of appropriate regulations and audit of antimicrobial use through hospital accreditation, inspection and national health insurance payment system. No action today, no cure tomorrow. Taiwan CDC would take a multifaceted, evidence-based approach and make every effort to combat antimicrobial resistance with stakeholders to limit the spread of multi-drug resistant strains and to reduce the generation of antibiotic resistant bacteria in Taiwan.     

Bacteriophage Polysaccharide Depolymerases and Biomedical Applications

Polysaccharide depolymerase, a polysaccharide hydrolase encoded by bacteriophages (or ‘phages’), can specifically degrade the macromolecule carbohydrates of the host bacterial envelope. This enzyme assists the bacteriophage in adsorbing, invading, and disintegrating the host bacteria. Polysaccharide depolymerase activity continues even within biofilms. This effectiveness means phages are promising candidates for novel antibiotic scaffolds. A comprehensive compendium of bacteriophage polysaccharide depolymerases has been compiled, together with their potential biomedical applications, such as novel antibiotics, adjuvants for antibiotics, bacterial biofilm disruptants, and diagnostic kits.     

Microbial biofilms in the gut: visualization by electron microscopy and by acridine orange staining

The expression of colonization factors by gut bacteria, the growth rate of gut bacteria, and the rate of plasmid exchange by gut bacteria indicate that biofilms are a normal component of bacterial growth in the large bowel. Further, in vitro experiments demonstrate that growth of normal enteric bacteria in biofilms can be facilitated by secretory IgA (SIgA) and by mucins, 2 major components of the gut milieu. However, biofilms have not been previously observed in the normal gut. In this study, bacterial colonies characteristic of biofilms were observed by electron microscopy in normal rat, baboon, and human gut by electron microscopy. Confirming these results, acridine orange staining of flash-frozen tissues revealed biofilms in the mucus lining along normal gut epithelium. Immunofluorescenct microscopy supported this finding and demonstrated an association between IgA and the biofilms. These findings provide direct evidence that biofilms are present and may play an important role in the commensal relationship between enteric bacteria and their hosts. Hematoxylin and eosin staining of formalin-fixed tissues resulted in dissociation of the luminal contents from the epithelium, suggesting that the association between biofilms and the gut epithelium is sensitive to some conditions used to preserve tissue for histologic evaluation.     

Quorum sensing : a novel target for the treatment of biofilm infections

Present-day treatment of chronic infections is based on compounds that aim to kill or inhibit growth of bacteria. Two problems are recognised to be intrinsically associated with this approach: (i) the frequently observed development of resistance to antimicrobial compounds; and (ii) the fact that all therapeutics are considerably less effective on bacteria growing as biofilms when compared with planktonic cells. The latter point is of particular importance as evidence has accumulated over the past few years that most chronic bacterial infections involve biofilms. The discovery of bacterial communication systems (quorum sensing systems) in Gram-negative bacteria which are believed to orchestrate important temporal events during the infectious process, including the production of virulence factors and the formation of biofilms, has afforded a novel opportunity to control the activity of infecting bacteria by other means than interfering with growth. Compounds that interfere with communication systems are present in nature. Such compounds should not only specifically attenuate the production of virulence factors but should also affect biofilm formation in a manner that is unlikely to pose a selective pressure for the development of resistant mutants.     

The effect of ultrasonic frequency upon enhanced killing ofP. aeruginosa biofilms

It is widely recognized that the bacteria sequestered in a biofilm on a medical implant are much more resistant to antibiotics than their planktonic counterparts. Recent studies have shown that application of antibiotic along with low power ultrasound significantly increases the killing of planktonic bacteria by the antibiotic. Herein is reported a similar application of antibiotic and ultrasound to sessile bacteria in biofilms ofPseudomonas aeruginosa on a polyethylene substrate. Biofilm viability was measured after exposure to 12 μg/ml gentamicin sulfate and 10 mW/cm² ultrasound at frequencies of 70 kHz, 500 kHz, 2.25 MHz, and 10 MHz. The results indicate that a significantly greater fraction of the bacteria was killed by gentamicin when they were subjected to ultrasound. However, ultrasound by itself did not have any deleterious effect on the biofilm viability. In addition, lower-frequency insonation is significantly more effective than higher frequency in reducing bacterial viability within the biofilm. The possible mechanisms of synergistic action are discussed.     

Antibiotics from neglected bacterial sources

The current crop of antibiotics in clinical use are either natural products or their derivatives. However, the rise of a multitude of different antibiotic resistant human pathogens has meant that new antibiotics are urgently needed. Unfortunately, the search for new antibiotics from traditional bacterial sources often results in a high rediscovery rate of known compounds and a low chance of identifying truly novel chemical entities. To overcome this, previously unexplored (or under investigated) bacterial sources are being tapped for their potential to produce novel compounds with new activities. Here, we review a number of antibiotic compounds identified from bacteria of the genera Burkholderia, Clostridium, Lysobacter, Pantoea and Xenorhabdus and describe the potential of organisms and their associated metabolites in future drug discovery efforts.     

Single and combination antibiotic susceptibilities of planktonic,adherent, and biofilm-grown Pseudomonas aeruginosa isolates cultured from sputa of adults with cystic fibrosis

Evidence suggests that Pseudomonas aeruginosa bacteria form biofilms within the airways of adults with cystic fibrosis (CF). The objective of this study was to determine whether clinical isolates of P. aeruginosa recovered from adults with CF have similar susceptibilities to individual antibiotics and to antibiotic combinations when grown as adherent monolayers or as biofilms compared to when they are grown using planktonic methods. Twelve multiresistant P. aeruginosa isolates, one mucoid and one nonmucoid from each of six CF patients, were grown conventionally under planktonic conditions, as adherent bacterial monolayers, and as biofilms. Each bacterial isolate remained genotypically identical despite being cultured under planktonic, adherent, or biofilm growth conditions. Isolates grown as adherent monolayers and as biofilms were less susceptible to bactericidal killing by individual antibiotics compared to those grown planktonically. More importantly, biofilm-grown bacteria, but not adherent monolayer-grown bacteria, were significantly less susceptible to two- and three-drug combinations of antibiotics than were planktonically grown bacteria (P = 0.005). We conclude that biofilm-grown bacteria derived from patients with CF show decreased susceptibility to the bactericidal effects of antibiotic combinations than do adherent and planktonically grown bacteria.     

Cell-wall-inhibiting antibiotic combinations with activity against multidrug-resistant Klebsiella pneumoniae and Escherichia coli

The increasing prevalence of hospital and community-acquired infections caused by multidrug-resistant (MDR) bacterial pathogens is rapidly limiting the options for effective antibiotic therapy. Systematic studies on combinations of already available antibiotics that could provide an effective treatment against MDR bacteria are needed. We tested combinations of antibiotics that target one important physiological function (peptidoglycan synthesis) at several steps, and studied Enterobacteriaceae (Klebsiella pneumoniae and Escherichia coli) for which multidrug resistance associated with ESBL-producing plasmids has become a major problem. To measure the effectiveness of antibiotics alone and in combination, we used checkerboard assays, static antibiotic concentration time-kill assays, and an improved in-vitro kinetic model that simulates human pharmacokinetics of multiple simultaneously administered antibiotics. The target strains included an MDR K. pneumoniae isolate responsible for a recent major hospital outbreak. A double combination (fosfomycin and aztreonam) and a triple combination (fosfomycin, aztreonam and mecillinam) were both highly effective in reducing bacterial populations in all assays, including the in vitro kinetic model. These combinations were effective even though each of the MDR strains was resistant to aztreonam alone. Our results provide an initial validation of the potential usefulness of a combination of antibiotics targeting peptidoglycan synthesis in the treatment of MDR Gram-negative bacteria. We suggest that a combination of fosfomycin with aztreonam could become a useful treatment option for such infections and should be further studied.     

Interactions among strategies associated with bacterial infection: pathogenicity,epidemicity, and antibiotic resistance

Infections have been the major cause of disease throughout the history of human populations. With the introduction of antibiotics, it was thought that this problem should disappear. However, bacteria have been able to evolve to become antibiotic resistant. Nowadays, a proficient pathogen must be virulent, epidemic, and resistant to antibiotics. Analysis of the interplay among these features of bacterial populations is needed to predict the future of infectious diseases. In this regard, we have reviewed the genetic linkage of antibiotic resistance and bacterial virulence in the same genetic determinants as well as the cross talk between antibiotic resistance and virulence regulatory circuits with the aim of understanding the effect of acquisition of resistance on bacterial virulence. We also discuss the possibility that antibiotic resistance and bacterial virulence might prevail as linked phenotypes in the future. The novel situation brought about by the worldwide use of antibiotics is undoubtedly changing bacterial populations. These changes might alter the properties of not only bacterial pathogens, but also the normal host microbiota. The evolutionary consequences of the release of antibiotics into the environment are largely unknown, but most probably restoration of the microbiota from the preantibiotic era is beyond our current abilities.     

Anti-biofilm efficacy of silver nanoparticles against MRSA and MRSE isolated from wounds in a tertiary care hospital

Purpose: Different approaches have been used for preventing biofilm-related infections in health care settings. Many of these methods have their own de-merits, which include chemical-based complications; emergent antibiotic resistant strains, etc. The formation of biofilm is the hallmark characteristic of Staphylococcus aureus and S. epidermidis infection, which consists of multiple layers of bacteria encased within an exopolysachharide glycocalyx. Nanotechnology may provide the answer to penetrate such biofilms and reduce biofilm formation. Therefore, the aim of present study was to demonstrate the biofilm formation by methicillin resistance S. aureus (MRSA) and methicillin resistance S. epidermidis (MRSE) isolated from wounds by direct visualisation applying tissue culture plate, tube and Congo Red Agar methods. Materials and Methods: The anti-biofilm activity of AgNPs was investigated by Congo Red, scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) techniques. Results: The minimum inhibitory concentration (MIC) was found to be in the range of 11.25-45 μg/ml. The AgNPs coated surfaces effectively restricted biofilm formation of the tested bacteria. Double fluorescent staining (propidium iodide staining to detect bacterial cells and fluorescein isothiocyanate concanavalin A (Con A-FITC) staining to detect the exopolysachharides matrix) technique using CLSM provides the visual evidence that AgNPs arrested the bacterial growth and prevent the glycocalyx formation. In our study, we could demonstrate the complete anti-biofilm activity AgNPs at a concentration as low as 50 μg/ml. Conclusions: Our findings suggested that AgNPs can be exploited towards the development of potential anti-bacterial coatings for various biomedical and environmental applications. In the near future, the AgNPs may play major role in the coating of medical devices and treatment of infections caused due to highly antibiotic resistant biofilm.     

Spiroplasma spp. biofilm formation is instrumental for their role in the pathogenesis of plant, insect and animal diseases

Spiroplasma spp. are important phyto and insect pathogens, and candidate causal agent/s of transmissible spongiform encephalopathies (TSE) in man and animals. These filterable wall-less bacteria are widely distributed in nature with an unspecified environmental reservoir. In this study we showed by scanning electron microscopy that spiroplasma form biofilm on an assortment of hard surfaces including mica, nickel and stainless steel. Spiroplasma were stuck to the surfaces by fibrillar threads consistent with curli fibers (an amyloid protein found in bacterial biofilms). After a lengthy time in cultures (6 weeks), spiroplasma in biofilm bound to mica disks lost their spiral shapes and formed coccoid forms interconnected by long (>2 μm) branched membranous nanotubules, therein representing direct conjugate connections between the cells. The affinity of spiroplasma biofilms for mica and nickel, and the membrane communications suggest that soil could be a reservoir for these bacteria. The persistence of clay bound spiroplasma in soil could serve as the mechanism of lateral spread of TSEs by ingestion of soil by ruminants. Spiroplasma binding to stainless steel wire supports bacterial contamination of surgical instruments following surgery on dementia patients as a mechanism of iatrogenic transmission of TSEs, especially with resistance of spiroplasma in biofilms to drying or exposure to 50% glutaraldehyde. The discovery of biofilm formation by spiroplasma addresses questions regarding environmental persistence of these organisms in nature and suggests novel mechanisms of intercellular communication and transmission.     

神经内科重症监护病房医院获得性肺炎 病原菌分布及耐药性分析

目的 探讨神经内科重症监护病房医院获得性肺炎的病原菌分布以及对抗生素的耐药性,为抗感染治疗提供参考。方法 2016年1月~2017年12月入住湖南省第二人民医院神经内科重症监护病房合并院内获得性肺炎的患者79例,采集痰标本进行细菌分离及培养,并对其进行相关药敏试验。结果 共分离出89株病原菌,其中革兰氏阴性菌占87.64%,革兰氏阳性菌10.11%,真菌2.25%。病原菌以鲍曼不动杆菌（24.72%）、肺炎克雷伯菌（19.10%）、铜绿假单胞菌（17.98%）,金黄色葡萄球菌为主（8.99%）为主;鲍曼不动杆菌对大部分抗菌药物耐药,铜绿假单胞菌对头孢哌酮舒巴坦和头孢他啶的耐药率较高,肺炎克雷伯菌耐药率低。金黄色葡萄球菌对青霉素耐药率高。结论 HAP致病菌以革兰氏阴性菌为主,其中鲍曼不动杆菌耐药严重,同时也应注意革兰氏阳性菌和真菌感染。     

Iberian wolf as a reservoir of extended-spectrum β-lactamase-producing Escherichia coli of the TEM, SHV, and CTX-M groups

The intensive use of antibiotics in human and veterinary medicine, associated with mechanisms of bacterial genetic transfer, caused a selective pressure that contributed to the dissemination of antimicrobial resistance in different bacteria groups and throughout different ecosystems. Iberian wolf, due to his predatory and wild nature, may serve as an important indicator of environmental contamination with antimicrobial resistant bacteria. The aim of this study was to characterize the diversity of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli isolates within the fecal microbiota of Iberian wolf. Additionally, the identification of other associated resistance genes, phylogenetic groups, and the detection of virulence determinants were also focused on in this study. From 2008 to 2009, 237 fecal samples from Iberian wolf were collected in Portugal. E. coli isolates with TEM-52, SHV-12, CTX-M-1, and CTX-M-14-type ESBLs were detected in 13 of these samples (5.5%). This study reveals the presence of ESBL-producing E. coli isolates, in a wild ecosystem, which could be disseminated through the environment. Moreover, the presence of resistant genes in integrons and the existence of virulence determinants were shown. The association between antibiotic resistance and virulence determinants should be monitored, as it constitutes a serious public health problem.     

An in vitro model of bacterial infections in wounds and other soft tissues

Werthén M, Henriksson L, Jensen PØ, Sternberg C, Givskov M, Bjarnsholt T. An in vitro model of bacterial infections in wounds and other soft tissues. APMIS 2010; 118: 156–64. There is growing evidence that bacteria play a crucial role in the persistence of chronic wounds. These bacteria are most probably present in polymer‐embedded aggregates that represent the biofilm mode of growth. Much work has been carried out to study the development of biofilms in vitro, in particular in attachment to solid surfaces. The observations from the chronic wounds indicate that the bacteria are not attached to a solid surface. Consequently, a new in vitro model is required to investigate biofilms in more wound‐like settings. This study describes such a novel in vitro model, with bacteria growing as biofilm aggregates in a collagen gel matrix with serum protein mimicking the wound bed of chronic wounds. The model was verified to comprise important hallmarks of biofilms such as the bacterial embedment in a matrix and increased antibiotic tolerance. Furthermore, we have verified the relevance of the model by comparing the organization of the bacteria in the model with the organization of the bacteria in a real chronic wound. We believe that we have developed an important new model for investigating bacterial biofilms in chronic wounds. This model may be used to study biofilm development in chronic wounds and to develop novel diagnostic tools as well as treatment strategies.     

The role of integrons in antibiotic resistance gene capture

Although recently discovered, integrons have played a primordial role in the evolution of bacterial genomes. They are best known as the genetic agents responsible for the capture and spread of antibiotic resistance determinants among diverse Gram-negative clinical isolates, and this activity is at the root of the antibiotic resistance phenomenon that has evolved over the last 60 years. The discovery of the ancestral chromosomal super-integrons, novel integron classes, and the multitude of gene cassettes they propagate solidify the crucial role of this system in adaptive bacterial evolution. Recent evidence suggests that evolutionarily old genetic recombination mechanisms for gene transfer have been adapted to the new antibiotic environment due to the heavy selective pressure of liberal antibiotic use in human medicine and animal husbandry.     

The phagosomal environment protects virulent Mycobacterium avium from killing and destruction by clarithromycin.

Murine bone marrow-derived macrophages (Mphis) infected with virulent strains of Mycobacterium avium (TMC 724 and a human clinical isolate) or with an avirulent opaque variant that spontaneously dissociates from the virulent human clinical isolate were subjected to a prolonged and continuous treatment with clarithromycin added at the MIC. The efficiency of this antibiotic in terms of inhibition of bacterial growth and bacterial degradation was evaluated during a 21-day treatment period. Growth was assessed by determination of CFU of intracellular bacteria and by a quantitative ultrastructural analysis which allowed us also to determine the extent of bacterial degradation. A similar treatment was applied to the same strains growing in liquid medium. Our data show that in liquid medium, clarithromycin caused a 90% decrease in CFU within 7 days of treatment. When applied to Mphis infected with virulent M. avium, clarithromycin immediately arrested bacterial growth but was unable to fully kill and degrade intracellularly growing virulent bacteria. After 21 days of treatment, 25% of intracellular bacteria were still morphologically intact. These bacteria resumed growth upon removal of the antibiotic, with a normal replication rate. These bacteria had not become more resistant to the drug, since the MIC was unchanged as compared to the one determined for the initial stock used to infect Mphis. Our data therefore suggest that the intraphagosomal environment protects bacteria from degradation. We propose that the inability of the drug to completely destroy bacteria is the result of a limited accessibility of the drug due to prevention of fusions between the immature phagosomes in which virulent bacteria reside and lysosomes in which clarithromycin accumulates. In accord with our proposal, we show that the avirulent opaque variant, which does not prevent phagosome-lysosome fusions (unpublished data), is finally destroyed by clarithromycin even within the phagosomal environment.     

The Calgary Biofilm Device: New Technology for Rapid Determination of Antibiotic Susceptibilities of Bacterial Biofilms

Determination of the MIC, based on the activities of antibiotics against planktonic bacteria, is the standard assay for antibiotic susceptibility testing. Adherent bacterial populations (biofilms) present with an innate lack of antibiotic susceptibility not seen in the same bacteria grown as planktonic populations. The Calgary Biofilm Device (CBD) is described as a new technology for the rapid and reproducible assay of biofilm susceptibilities to antibiotics. The CBD produces 96 equivalent biofilms for the assay of antibiotic susceptibilities by the standard 96-well technology. Biofilm formation was followed by quantitative microbiology and scanning electron microscopy. Susceptibility to a standard group of antibiotics was determined for National Committee for Clinical Laboratory Standards (NCCLS) reference strains: Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, and Staphylococcus aureus ATCC 29213. Growth curves demonstrated that biofilms of a predetermined size could be formed on the CBD at specific time points and, furthermore, that no significant difference (P > 0.1) was seen between biofilms formed on each of the 96 pegs. The antibiotic susceptibilities for planktonic populations obtained by the NCCLS method or from the CBD were similar. Minimal biofilm eradication concentrations, derived by using the CBD, demonstrated that for biofilms of the same organisms, 100 to 1,000 times the concentration of a certain antibiotic were often required for the antibiotic to be effective, while other antibiotics were found to be effective at the MICs. The CBD offers a new technology for the rational selection of antibiotics effective against microbial biofilms and for the screening of new effective antibiotic compounds.